In 2001/2002 the groups of Sharpless and Meldal independently defined the concept of “Click chemistry” and the criteria for a transformation to be considered a “Click” reaction [2] and [3]. Since then, the copper catalysed reaction of azides with alkynes to give 1,2,3-triazoles (the 1,3-dipolar Huisgen cycloaddition[1]) has become the most widely used Click reaction. As a result of its mild conditions and high efficiency, this reaction has found a myriad of applications in biology and materials science, such as DNA labelling purposes [16].
One of the limitations of the method when applied to biomolecule labelling concerns the use of Cu(I) as catalyst of the Click reaction. Cu-ions are toxic to bacterial and mammalian cells, thus limiting the use of reaction inside living systems. Furthermore, for DNA, and RNA, improper handling of the catalyst can cause degradation of the oligonucleotides due to Cu-ion catalyzed phosphordiester hydrolysis [17, 18]. Solutions to those problems have been reported and include the in situ Cu(I) generation and/or the use of Cu(I)-stabilizing ligands. The latter is limited to substrates and experimental conditions, which tolerate the presence of organic solvents involved in the Click reaction in order to dissolve the organic ligand (generally a tertiary amine). Molecules such as proteins or long DNA strands eventually precipitate or form insoluble agglomerates upon the addition of even small amounts of organic solvents thus negatively affecting the outcome of the Click reaction.
In any case free Cu(I) ions have a limited life time under ambient conditions, being oxidised to the Click reaction-inactive Cu(II). Therefore, the freshly prepared Cu(I) ligand mixture for the Click reaction needs to be handled carefully and rapidly. Therefore this procedure can only be used in labor-intense manual work, but not in automated process.
According to recent reviews [4], a Click reaction in the presence of copper(II) sulfate (ca. 1%) and sodium ascorbate (ca. 10%) serves to generate catalytically active Cu(I) in situ in an aqueous medium (e.g. H2O/tBuOH) and is typically preferred. Alternative conditions, such as in situ oxidation of Cu(0) or direct introduction of Cu(I) salts (usually CuI or CuBr), have also been used [5]. While both reaction partners have been individually coupled under solid phase conditions, (e.g. on polystyrene) [3, 6], examples of Click chemistry mediated by a source of heterogeneous copper(I) are rare. One report relies on chelation to potentially labile copper by a polystyryl-based benzylic amine [7a]. Only unhindered, low-molecular-weight, and non-basic nitrogen-containing examples were studied therein, without data quantifying losses of copper from the solid support. Other studies are also limited, based on suspensions of unsupported copper clusters [7b].
Lipshutz et al. recently described the virtues of copper-in-charcoal (Cu—C) as a simple, inexpensive, and especially general and efficient heterogeneous catalyst for use in this emerging area [19]. Impregnation of activated wood charcoal (Aldrich, 100 mesh, $53.90/kilo)[8] with a source of Cu(II), e.g. Cu(NO3)2 in water using an ultrasonic bath was described to lead, after distillation of water and drying, to nanoparticle-sized Cu(I)—C [9]. The Cu(I) is generated from Cu(II) not in an in situ reduction, but via a charcoal-mediated reduction, which allows pre-assembling and storage of the so-generated Cu(I) source for extended time. As both CuO and Cu2O have been proposed as the species present within a charcoal matrix [10], the presence of Cu(I) suggested that a reducing agent might not be needed. Indeed, the authors have shown that upon mixing benzyl azide with phenylacetylene (1:1) in dioxane at room temperature in the presence of 10 mol % Cu—C, cycloaddition was complete within 10 hours. Filtration and solvent evaporation afforded pure triazole regiospecifically and near-quantitatively [19]. Furthermore, Lipshutz et al. demonstrated the absence of free Cu(I) in solution, corroborating the heterogeneous nature of the Click-reaction event.
In conclusion, Lipshutz et al. reported that highly efficient Click chemistry between organic azides and terminal alkynes can be heterogeneously catalyzed by copper nanoparticles mounted within the pores of activated charcoal [9]. Furthermore, the authors have shown that reactions can be accelerated with stoichiometric Et3N or by simply increasing the reaction temperature. Under microwave irradiation, triazoles can be formed in minutes at 150° C. Cycloadditions can be carried out in a purely organic medium, in aqueous solvent mixtures, or in pure water. Solubility issues, copper contamination, and modest yields usually associated with the choice of copper salt are completely averted. External ligands known to accelerate Click reactions are not needed. The catalyst appears to be unaffected by exposure to air, suggesting a substantial shelf life. Steric congestion in one or both partners is well tolerated, and product isolation is notably facile, as Lipshutz et al. [19] demonstrated.
The use of Cu/C catalysts for Click reactions with organic molecules in free form is also disclosed in [20] and [21]. None of these documents, however, describes a Click reaction wherein one of the Click partners is immobilized on a solid carrier.
Click reactions involving the use of biomolecules are disclosed in [16], [22], [23] and [24]. These reactions are performed in the presence of a soluble catalyst.
The present invention describes the application of heterogeneous catalysts, particularly Cu(I)—C-catalysts for the labelling of biomolecules such as DNA and many possible easy-to-use devices or methods based on this invention, several of which are shown in this application as proof-of-concept and example.